Pharmaceutical Formulation for Regulating the Timed Release of Biologically Active Compounds Based on a Polymer Matrix

ABSTRACT

A formulation containing a biologically active compound having a structure with hydrogen bonding sites is blended with a polymer having a structure with complementary hydrogen bonding sites, the polymer forming hydrolytic degradation products that promote the release of the biologically active compound from the polymer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/169,410, filed Jul. 1, 2002, now U.S. Pat. No. 7,521,061, which is aNational Stage of International Application No. PCT/US01/00030, filedJan. 2, 2001, which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 60/174,137, filed Dec. 31, 1999, thedisclosures of all three of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a new approach to the delayed or pulsedrelease of biologically active compounds having pharmaceutical activity,particularly peptides such as INTEGRILIN™, from a polymer matrix. Inthis system no complicated barrier mechanism is required to prevent therelease of the peptide during the lag time, a high loading of thewater-soluble peptide is readily achieved, and the length of the delayof the release of the peptide is easily controlled.

Previously, limited release of INTEGRILIN™ was reported from poly(DTHadipate), a member of the tyrosine-derived polyarylates, despite highloadings of the peptide (30% w/w). Subsequent investigations indicatedthat interactions between the peptide and the polymer were responsiblefor the minimal release (˜5% of the loaded peptide). Since hydrogenbonding was a component of the interactions, the release of the peptidefrom poly(DTH adipate) was demonstrated to be sensitive to the pH withinthe polymer matrix.

The literature is replete with examples of the delayed or pulsed releaseof active agents using polymeric materials. However, it is possible todivide these systems into two basic categories; those that depend on anenvironmental stimulus to induce release of the active agent from thepolymeric matrix and those that are designed to release the drug afterparticular intervals of time have elapsed. Examples of environmentalstimuli that have been used for this application are electricalimpulses, pH or temperature changes, application of magnetic fields, orultrasound.

Those systems that are time-controlled can further be divided into thosethat use a barrier technology that is placed around the active agentthat is designed to degrade or dissolve after a certain time interval,and those that use the degradation of the polymer itself to induce therelease of the active agent.

One approach of this category has been to prepare a polymeric hydrogelcomposed of derivitized dextran and to incorporate into the hydrogel, amodel protein, I_(g)G, with an enzyme, endo-dextranase, that degradesthe hydrogel. It was observed that without the enzyme the release of theprotein was very slow. However, when the enzyme was included in theformulation, the release rate was dependent on the concentration of theenzyme. At high concentrations, the release was fast and complete. Atlow concentrations, the release was delayed.

A correlation was found between the delay time and the rate of thedegradation of the hydrogel. The interpretation of the data was that themesh size of the hydrogel was too small for efficient diffusion of alarge protein molecule such as I_(g)G, but as the enzyme degraded thepolymer, the mesh size increased and diffusion was unimpeded.

Delayed release in association with hydrolytic degradation of thepolymer has also been investigated. Heller's poly(ortho esters) areviscous ointments at room temperature and when mixed with a modelprotein, lysozyme, demonstrated a delayed release profile. The length ofthe delay time was found to correlate with polymer molecular weight andalkyl substituent of the polymer. These experiments, however, arelimited by the fact that all of the drug release experiments wereconducted at room temperature, perhaps, because the polymers are viscousat room temperature, but not at the physiological temperature of 37° C.

Ivermectin, a water insoluble antiparasitic agent for veterinaryapplications, was encapsulated in PLGA (50:50) microspheres and thesubsequent pulsed release of this agent, in vivo, was shown to bedependant on the degradation rate of the polymer matrix. Pulsed anddelayed release of active agents from PLGA microspheres was mostintensely studied by Cleland et al. The PLA or PLGA microspheres areprocessed using a high kinematic viscosity of polymer solution and ahigh ratio of polymer to aqueous solution. This produces densemicrospheres, which require sever bulk erosion to release the drug.These conditions yield microspheres that have low loading (generally 1%w/w), moderate bursts, and lag times during which significant leachingof drug occurs.

SUMMARY OF THE INVENTION

The technology described in this disclosure represents a departure fromthe prior art. In this system, bonding interactions between a polymerand an active compound are used to inhibit the release of the activecompound, and the polymeric degradation products are used to control thelength of time preceding release of the active compound. The bondinginteractions are composed of hydrogen bonding and hydrophobic forces anddevelop when a highly functional polymer is employed.

Therefore, according to one aspect of the present invention, aformulation containing a biologically active compound is provided,having a structure with hydrogen bonding sites, blended with a polymerhaving a structure with complementary hydrogen bonding sites, thepolymer forming hydrolytic degradation products that promote the releaseof the biologically active compound from the polymer.

The formulation thus consists of two components, a polymer and an activecompound blended together. The present invention thus provides aformulation system that uses the degradation products of selectedpolymers to trigger the release of the active compound from the matrixof the polymer. Using this method, active compounds can be very simplyformulated with the polymer and be programmed to be released at desiredintervals, requiring no sophisticated barriers to prevent the prematurerelease of the active agent.

There are many drugs that are more effective when given to the patientin a pulsatile manner as opposed to a continuous release fashion. Forexample, an area of great interest, currently, for this type of deliverysystem is single-shot immunity. Immunity is best induced by a pulsatiledelivery of the agent, hence the need for booster shots. It has beensuggested that it would be more economical and effective, especially inthird world countries, if a tetanus toxoid or gp120 (under developmentfor an AIDS vaccine) could be implanted once into the patient and theboosters be automatic and preprogrammed from the implanted or injecteddevice.

Therefore, the present invention also includes a method for thepulsatile delivery of a biologically active compound to a patient inneed thereof comprising administering to the patient the formulation ofthe present invention.

This type of drug delivery is not only important for amino acid baseddrugs but also for hormonal based drug delivery. Fertility and birthcontrol drug therapy for both animals and humans is not continuous, butrather cyclic in nature since these therapies work synergistically withthe menstrual cycle and the corresponding hormonal flux. This is anotherdirection in drug delivery in which this type of delayed pulsed releaseof an active agent would be applicable.

Agricultural applications which require the timed dosing of fertilizers,weed-killers, and other active agents is another area where thisinvention would be important.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of tyrosine-derived polyarylates;

FIG. 2 depicts the amino acid sequence of INTEGRILIN™;

FIG. 3 depicts release from poly(DTH adipate) films containing 30% (w/w)peptide;

FIG. 4 depicts release from D,L-PLA and poly(ε-caprolactone) filmscontaining 30% (w/w) peptide;

FIG. 5 depicts percent mass retention of poly(DTH adipate) samplescontaining 30% (w/w) peptide;

FIG. 6 depicts percent mass retention data for D,L-PLA samplescontaining 30% (w/w) peptide;

FIG. 7 depicts percent water absorption by films of PCL and PLAcontaining 30% (w/w) peptide;

FIG. 8 depicts percent water absorption by films of poly(DTH adipate)both with and without peptide;

FIG. 9 depicts percent molecular weight retention of neat poly(DTHadipate) samples to that of poly(DTH adipate) containing 30% (w/w)peptide, and to that of 10% PEG/90% poly(DTH adipate);

FIG. 10 depicts the effect of ionic strength on the release of 30% (w/w)INTEGRILIN™ from poly(DTH adipate) films;

FIG. 11 depicts release from poly(DTH adipate) films containing 30%(w/w) peptide at pH 2.2 without added electrolytes;

FIG. 12 depicts water uptake of poly(DTH adipate) films containing 30%(w/w) peptide at pH 2.2 without added electrolytes;

FIG. 13 depicts hydrogen bonding between a tyrosine-derived polyarylateand a peptide;

FIG. 14 depicts the chemical structure of poly(DTH dioxaoctanedioate);

FIG. 15 depicts release of peptide from poly(DTH dioxaoctanedioate);

FIG. 16 depicts the chemical structure of poly(DTE carbonate);

FIG. 17 depicts release of peptide from poly(DTE carbonate) samplescontaining 15% (w/w) peptide;

FIG. 18 depicts the chemical structure of desaminotyrosyltyrosine (DT);

FIG. 19 depicts percent molecular weight retention of neatpoly(DT-co-DTH adipate) films with 0, 5, 10, 15 mole percent of DT;

FIG. 20 depicts percent water uptake of neat poly(DT-co-DTH adipate)films with 0, 5, 10, and 15 mole percent DT;

FIG. 21 depicts release of peptide from poly(DT-co-DTH adipate)matrices;

FIG. 22 depicts release of peptide from 30% (w/w) poly(DT-co-DTHadipate) films;

FIG. 23 depicts pH measurements of buffer of samples of poly(DT-co-DTHadipate) with 15% (w/w) peptide; and

FIG. 24 depicts percent molecular weight retention of samples ofpoly(DTH adipate) containing various percentages of DT incubated in PBSat 37° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In its broadest embodiment, polymers that are suitable for use in thepresent invention are any polymer that contains hydrogen-bonding sitesas part of its structure and degrades to form products that promote therelease of a biologically active compound from the polymer.Biocompatible polymers are required for biomaterial end-useapplications.

Preferred polymers are copolymers containing a hydrophilic monomer and ahydrophobic monomer. In a more preferred embodiment, the copolymer isselected from the tyrosine-derived polyarylate libraries disclosed in WO99/24107 and WO 99/52962, the disclosures of both of which areincorporated herein by reference. The copolymers of WO 99/24107 containa hydrophilic monomer with a pendant carboxylic acid group,desaminotyrosyltyrosine, which degrades to form acidic degradationproducts. The other monomer, a desaminotyrosyltyrosine ester, alsocontains hydrogen bonding sites for retention of the active compound. Awater soluble yet hydrophobic dicarboxylate monomer forms polyarylatelinkages between the two diols.

Members of the tyrosine-derived polyarylate library all share the samehighly functional structural template but are distinguished from oneanother by subtle structural changes. The functional groups of the maintemplate provide sites for interactions. These are pi stacking of itsaromatic rings with an aromatic ring of a peptide, or hydrogen bondingof the α-amino carboxylate region with a corresponding group in thepeptide. The small structural variations between members allow thefine-tuning of these interactions to suit particular proteins orpeptides.

Also preferred are any of the copolymers that can be derived from thetyrosine-derived diphenol compounds of U.S. Pat. No. 5,587,507 and thetyrosine-derived dihydroxy monomers of WO 98/36013, the disclosures ofboth of which are also incorporated herein by reference, using theprocess of WO 99/24107 for forming free carboxylic acid moieties. Inaddition to the above-referenced polyarylates, examples include thepolycarbonates of U.S. Pat. No. 5,099,060, the polyiminocarbonates ofU.S. Pat. No. 4,980,449, the polyphosphazenes and polyphosphates of U.S.Pat. No. 5,912,225, polyurethanes, including the polyurethanes of U.S.Pat. No. 5,242,997, the random poly(alkylene oxide) block copolymers ofU.S. Pat. No. 5,658,995, and a wide range of other polymers that can bederived from the above-referenced tyrosine-derived diphenol compounds,the tyrosine-derived dihydroxy compounds and similar peptides. All ofthe above-referenced patent publications are incorporated herein byreference. Notably, corresponding polymers of the tyrosine-deriveddihydroxy compounds can be made by any of the processes of any of theabove-referenced patents disclosing polymers of tyrosine-deriveddiphenol compounds.

A particularly preferred copolymer is the desaminotyrosyltyrosine (DT)copolymer of poly(desaminotyrosyltyrosine hexyl ester adipate) (Poly(DTHadipate)), depicted in FIG. 1 (y=4; R=hexyl). Poly(DT-CO-DTH adipates)having a weight-average molecular weight between about 80,000 and about200,000 daltons is particularly preferred.

In a preferred embodiment, the present invention uses pH sensitivity tocontrol the release of an active compound. It was discovered that theaccumulation of acidic polymer degradation residues in the matrix of apolymer/peptide blend weakened the interactions between the peptide andthe polymer so that the peptide could be released. An inversecorrelation was demonstrated between the mole percent of acid moietiesin a polymer and the length of the lag time preceding release indicatingthat timed release of a peptide, or any active agent with hydrogenbonding sites, can be controlled by mole percent of acid moieties in apolymer.

Any biologically active compound with hydrogen-bonding sites that can bephysically dispersed within the polymer can be used as an activecompound for release (e.g. FIG. 13). Examples of hydrogen bonding sitesinclude primary and secondary amines, hydroxyl groups, carboxylic acidand carboxylate groups, carbonyl (carboxyl) groups, and the like. Whileone can apply the current invention to any active compound that hashydrogen bonding sites, including natural and unnatural antibiotics,cytotoxic agents and oligonucleotides, amino acid derived drugs such aspeptides and proteins seem to be most appropriate for this technology.The compositions of the present invention overcome some of thedifficulties encountered in previous attempts to formulate controlledrelease devices that show reproducible release profiles without burstand/or lag effects. In its most preferred embodiment, the activecompound is a peptide that is stable under mildly acidic conditions.

Peptide drugs suitable for formulation with the compositions of thepresent invention include natural and unnatural peptides, oligopeptides,cyclic peptides, library generated oligopeptides, polypeptides andproteins, as well as peptide mimetics and partly-peptides. Peptide drugsof particular interest include platelet aggregation inhibiting (PAI)peptides, which are antagonists of the cell surface glycoproteinIib/IIIa, thus preventing platelet aggregation, and ultimately clotformation. Preferred PAI peptides include the PAI peptides disclosed byWO 90/15620, the disclosure of which is incorporated herein byreference, particularly INTEGRILIN™ (FIG. 2), a medically useful cyclicPAI heptapeptide.

The compositions of the present invention are suitable for applicationswhere localized drug delivery is desired, as well as in situations wheresystemic delivery is desired. Therapeutically effective dosages may bedetermined by either in vivo or in vitro methods. For each particularcompound of the present invention, individual determinations may be madeto determine the optimal dosage required. The range of therapeuticallyeffective dosages will naturally be influenced by the route ofadministration, the therapeutic objectives, and the condition of thepatient. For the various suitable routes of administration, theabsorption efficiency must be individually determined for each drug bymethods well known in pharmacology. Accordingly, it may be necessary forthe therapist to titer the dosage and modify the route of administrationas required to obtain the optimal therapeutic effect. The determinationof effective dosage levels, that is, the dosage levels necessary toachieve the desired result, will be within the ambit of one skilled inthe art. Typically, applications of compound are commenced at lowerdosage levels, with dosage levels being increased until the desiredeffect is achieved. The release rate of the drug from the formulationsof this invention are also varied within the routine skill in the art todetermine an advantageous profile, depending on the therapeuticconditions to be treated.

A typical dosage might range from about 0.001 mg/kg to about 1000 mg/kg,preferably from about 0.01 mg/kg to about 100 mg/kg, and more preferablyfro about 0.10 mg/kg to about 20 mg/kg. Advantageously, the compounds ofthis invention may be administered several times daily, and other dosageregimens may also be useful.

The compositions may be administered subcutaneously, intramuscularly,colonically, rectally, nasally, orally or intraperitoneally, employing avariety of dosage forms such as suppositories, implanted pellets orsmall cylinders, aerosols, oral dosage formulations and topicalformulations, such as ointments, drops and transdermal patches.Liposomal delivery systems may also be used, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.

The following non-limiting examples set forth herein below illustratecertain aspects of the invention. All parts and percentages are byweight unless otherwise noted and all temperatures are in degreesCelsius. The PAI peptide was obtained from COR Therapeutics of South SanFrancisco, Calif., and used without further purification. Solvents wereof “HPLC grade” and were obtained from Fisher Scientific of Pittsburgh,Pa.

EXAMPLES

INTEGRILIN™ (antithrombotic injection) was chosen as the model peptideto explore the drug delivery applications of these materials (FIG. 2).This compound is a synthetic cyclic readily water-soluble heptapeptidewhich is a highly potent glycoprotein IIb/IIIa antagonist. This compoundhas successfully demonstrated antithrombogenic behavior in vivo anddevices fabricated by the formulation of this peptide into a polymermatrix with this property would have many useful cardiovascularapplications. In addition, this polymer contains an RGD sequence andtherefore a device containing this peptide could possibly findapplications as a component in scaffolds for tissue regeneration.

The blend of INTEGRILIN™ and poly(DTH adipate) was described in the U.S.Pat. No. 5,877,223, the disclosure of which is incorporated herein byreference. There, it was mentioned that formulating films from thesecomponents using the coprecipitation melt-press technique resulted inspecimens that released only trace amounts of peptide when incubated inPBS at 37° C. This was unexpected because the peptide is readilywater-soluble.

Polymer Synthesis and Specifications

Tyrosine derived polyarylates were synthesized as described in U.S. Pat.No. 5,877,224 and in WO 99/24107. Poly(DTH adipate) R=hexyl and y=4),was the polymer specifically chosen for this study though many of thephysical phenomena reported here for this polymer have been observedwith others in this polymer family. The polymers used had molecularweights ranging between 80-120 kDa. The particular polymers synthesizedwere poly(DT_(0.05)co-DTH_(0.95)adipate,Poly(DT_(0.10)-co-DTH_(0.09)adipate), and poly(DT_(0.15)-co-DTH_(0.85)adipate). The molecular weights of the polymers used ranged from 60-80kDa. D,L-PLA and poly(ε-caprolactone) were purchased from Medisorb andAldrich, respectively. Both were of molecular weight 100 kDa andfabricated into release devices in the same manner as the poly(DTHadipate).

Fabrication of Release Devices

The peptide was obtained from COR therapeutics, Inc. The peptide, asreceived, was 98-99% pure and used without further purification.Compression molded films were fabricated from a co-precipitatecontaining 30% peptide and 70% polymer by weight. This co-precipitatewas prepared by dissolving 0.15 g of peptide in 5 ml of methanol (HPLCgrade) and 0.35 g polymer in 5 ml of methylene chloride (HPLC grade) andmixing the two solutions together to form a clear solution. Thisresultant solution was added drop-wise into 100 ml of stirred ethylether maintained at −78° C. White spongy precipitates were formed,filtered using a sintered glass filter, and dried under vacuum. Afterdrying the co-precipitate was compression molded at 90° C. under apressure of 5,000 psi. Films with a thickness of 0.1 nm (±0.02 mm) wereobtained.

Device Characterization

Peptide loading was determined by dissolving 10.0 mg of a film in THF(HPLC grade) (1.0 ml) in a 10 ml volumetric flask and adding PBS(phosphate buffer saline) until the 10 ml line. The mixture was stirredfor a minimum of 6 hours followed by HPLC analysis of the drug contentin the aqueous medium. Methylene chloride replaced THF whencharacterizing samples composed of PLA or poly(ε-caprolactone) due totheir insolubility in THF.

Peptide Release Study

Films were cut into 0.5 cm² squares. The mean mass of the samples was 21mg (±5). Each specimen was individually placed into 20 ml glassscintillation vials containing 10 ml of phosphate buffered saline (pH7.4, 37° C.). The standard PBS solution used was composed of 10 mMphosphate buffer saline, 138 mM NaCl and 2.7 mM KCl. The buffer waschanged at each time point and analyzed by HPLC for release of thepeptide. There was a minimum of three samples per time point, eachsample originating from a different film. The HPLC method involved a 3cm C-18 Perkin Elmer cartridge column with a gradient mobile phase whichbegan at 80% water/20% acetonitrile and ended with 75% water during aperiod of 5 minutes at a flow rate of 1 ml/min. Both the acetonitrileand water contained 0.1% (v/v) trifluoroacetic acid. The column wascalibrated with known concentrations of the peptide dissolved in PBS toestablish a calibration curve and the INTEGRILIN™ contained in thebuffer of each sample was quantified using this curve. The HPLC pumpused was a Perkin Elmer Series 410 LC pump and the detector used was aPE LC-235 diode array UV-VIS detector set at 280 nm. The data collectedwas analyzed using a PE Nelson 3000 Series Chromatography Data System.

At designated times, the samples were removed, rinsed with deionizedwater, blotted with a Kimwipe tissue and either placed in a vial forsubsequent vacuum drying for mass retention and molecular weightretention studies or used for thermal gravimetric analysis (TGA) wateruptake studies. Those devices that were not needed for gel permeationchromatography (GPC) or TGS studies were dissolved in organic solventsubsequent to drying and the peptide content extracted to ensure thatall loaded peptide was accounted for.

Molecular Weight Determination of the Polymers Using GPC

The molecular weights of the poly(DTH adipate) samples were calculatedrelative to a set of monodispersed polystyrene standards (PolymerLaboratories, Ltd. Church Station, U.K.) without further corrections.The GPC chromatographic system consisted of a Waters 510 HPLC pump, aWaters 410 differential refractometer detector, and a Digital Venturi's466 PC running Millenium (Waters Corp.) software for data processing.Two PL-gel columns 30 cm in length (pore sizes of 10³ and 10⁵ Å, PolymerLaboratories Ltd.) operated in a series at a flow rate of 1 ml/min inTHF.

Samples composed of PLA or PCL were dissolved in methylene chlorideinstead of THF, but otherwise analyzed in the same way as the poly(DTHadipate) samples.

Water Content Determination Using Thermogravimetric Analysis (TGA)

A small sample (10 mg) was cut from a specimen and placed in an aluminumTGA pan. The sample was heated under a nitrogen flow at a rate of 10°C./min from room temperature to 225° C. The water uptake was measured bythe loss in weight of the sample as it was heated from room temperatureuntil 150° C.

Water Content Determination Using the Microbalance

At pre-determined time points, the samples were removed from the buffer,rinsed with deionized water and blotted dry. The sample's wet weight(W_(w)) was immediately taken using an electronic balance. The dryweight (W_(d)) was taken after the sample was dried under vacuum for atleast two weeks, by this time constant weight was achieved. The amountof water uptake was calculated from the following equation:

% Water uptake=[(W _(w) −W _(d))/W _(d)]×100  Eq. (2.1)

Differential Scanning Calorimetry Analysis (DSC) to Measure the MeltingPoint of the Peptide and Melt Transitions in the Polymer Film

DSC was used to determine the melting point of the peptide. A sample ofapproximately 2 mg of peptide was weighed out and sealed in a crimpedaluminum DSC pan. The sample was heated at 12° C./min from roomtemperature to 200° C., under nitrogen flow. DSC was also used todetermine whether there is a melting transition associated with thepolymer films that contain 30% (w/w) peptide. A sample size of 6 mg offilm was sealed in a crimpled aluminum DSC pan and heated at 12° C./minuntil 200° C., under nitrogen flow. Melting point of the sample wasdetermined by the temperature at which the sharp endotherm of meltingoccurred. All data was analyzed using the first-run thermogram. An emptyaluminum pan was used as a reference in each experiment. The instrumentused was a DSC 910 (TA instruments). The instrument was calibrated withindium (m.p.=156.61° C.) before use.

Percent Mass Retention Study

The percent mass retention of the samples was calculated in thefollowing manner. The sample was removed from the PBS incubation medium,rinsed in deionized water, and blotted with a Kimwipe tissue. It wasplaced in a fresh vial and dried under vacuum for 2 weeks. Followingthis dessication period, it was weighed (W_(d)). The mass obtainedfollowing incubation and drying was compared to the initial mass(W_(o)). The formula for calculating percent mass retention is thefollowing:

% Mass loss=[(W _(o) −W _(d))/W _(o)]×100  Eq. (2.2)

Fabrication and Incubation of Films Under Acidic Conditions

The same formulation protocol mentioned above was followed for thesefilms, with the exception that concentrated JCl (12 molar) was addeddrop-wise to the stirred peptide/methanol solution until the pH, asmeasured by a pH meter dropped from 6.8 to 2.

The acidic media for the in vitro incubation studies conducted at pH of2 was prepared in the following manner. Standard PBS solution was usedand 12 M HCl was added drop-wise into the PBS until the PBS until the pHmeter indicated that the desired pH had been obtained.

Incubation of Films Under Varying Ionic Strength Conditions

Three sets of films were prepared in the standard method mentionedabove, one set was incubated in HPLC water, used as is. Another wasincubated in standard PBS buffer. The last set was incubated in PBSbuffer that was twice the concentration of the standard PBS solution.

The Effect of the Peptide on the Glass Transition Temperature ofPoly(DTH Adipate)

The glass transition temperature of sets of films was measured usingDynamic Mechanical Analysis (DMA). Measurements were performed on a DMA983 from TA Instruments in a flexural bending deformation mode ofstrain. Each set of films contained a different weight percentage ofpeptide ranging from 0%-30% (w/w) of peptide. Samples of approximatesize 5×10×1 mm were cut from the films and mounted on the instrumentusing low mass clamps. The samples were cooled using a liquid nitrogencooling accessory to −30° C. The frequency was fixed at 1 Hz and theamplitude was 1 mm. The glass transition was read from the maxima of theE″ peak.

Reduction of the Peptide Using Dithiothreitol (DTT)

INTEGRILIN™ (4.12 mg) was placed in a 25 ml roundbottom flask. To thisflask was added 50.12 mg of dithiothreitol. A minimum of 20 moles of DTTwas required per disulfide bridge (this is 62 moles of DTT per disulfidebridge). Then 3 ml of water was added and flask was stoppered. Thecontents of flask were stirred with a magnetic stirrer. Every few hours,an aliquot of reaction mixture was removed from the flask, diluted withHPLC water, and analyzed with HPLC. As the reaction continued, the peakat 1.7 minutes corresponding to the intact peptide decreased and thepeak at 2.5 minutes corresponding to the peptide with the cleaveddisulfide bridge increased. Virtually all of the peptide had beenreduced after stirring overnight.

Following the conversion, the reaction mixture was lyophilizedovernight. In order to extract the reduced product, 5 ml of diethylether was added to dissolve the DTT and precipitate the peptide. Thismixture was stirred for 3 hours and the resulting suspension wasfiltered using filter paper. The filtered material was dried undervacuum overnight.

The presence of free SH groups was assayed in the following manner. Asaturated solution of lead acetate in ethanol was made and a fewmilliliters of this solution were poured into the vial containing thedried reduced peptide. A yellow color developed indicating the presenceof the lead sulfur complex: As a control, equal volume of this leadsolution was added to the peptide with the intact disulfide bridge andno yellow color was detected.

Evidence for and Investigation of Interactions Between the Peptide andPoly(DTH Adipate)

Formulation of INTEGRILIN™ with Poly(DTH Adipate)

Films composed of poly(DTH adipate) containing loadings of 5, 10, 15,20, and 30% (w/w) peptide were prepared. Films containing even thehighest loading were clear and flexible. In contrast, the films composedeither of D,L-PLA or poly(ε-caprolactone) (PCL) containing the same loadof peptide were opaque and brittle. The clarity of thepeptide/polyarylate films indicated that the phase separation in thecase of the peptide and poly(DTH adipate) was sufficiently reduced thatthe separate polymer and peptide domains were too small to scatterlight. This suggested an enhanced compatibility of peptide andtyrosine-derived polymer relative to the D,L-PLA or PCL and peptide.

The flexibility of the polyarylate films that contained peptide relativeto those composed of the peptide and either of aliphatic polyesters canbe explained by the lower glass transition temperature of thepolyarylate (37° C.) as compared to that of PLA (52° C.), and theamorphous nature of the polyarylate as compared to PCL.

Release of Peptide from Films Incubated at 37° C. and at pH=7.4

In this experiment the in vitro release behavior of the peptide, undersimulated physiological conditions, from various polymer matrices wasobserved. Both the aliphatic polymers released and the peptidecompletely within three hours. In contrast, the poly(DTH adipate)demonstrated only trace release, over a period of 77 days, under theidentical conditions (FIGS. 3 $ 4).

Percent Mass Retention of Incubated Samples

Poly(DTH adipate) samples containing 30% (w/w) peptide lost on average5% mass during the 77 day incubation period (FIG. 5). In contrast, theD,L-PLA samples that were formulated in the identical fashion as thepoly(DTH adipate) samples lost about 30% of their mass within two hours(FIG. 6). The results of these experiments, therefore, were consistentwith the data obtained from the HPLC. In the case of the poly(DTHadipate) films containing 30% (w/w) peptide, the HPLC data indicate thatthese films released less than 10% of the loaded peptide (FIG. 3). Thistranslates into a mass loss for the entire sample of about 3% over the77 day period. This is in agreement with the average 5% mass lossobserved for these samples.

In contrast to the poly(DTH adipate) samples that demonstrated minimalmass loss, the PLA samples showed extensive mass loss. These filmsamples also contained 30% (w/w) peptide. HPLC data indicated that thesesamples released all of the peptide that they contained, this translatesinto a 30% mass loss over the three hour incubation period. Theresulting percent mass retention data is about 70% for these samples istherefore in agreement with the HPLC results. Furthermore, since thepeptide was released so rapidly by the PLA and PCL matrices, it can beconcluded that the peptide is small enough to readily diffuse throughthe polymer chains and the development of pore structures andinterconnecting channels is not necessary to release the molecules ofpeptide that are deep within the film. Therefore there should be minimalimpedance for release of the peptide from the polyarylate.

Measurement of Water Absorption by Polymer Films During Incubation

Specimens of poly(DTH adipate) containing 30% (w/w) peptide absorbedabout 10% by weight water within the first day and maintained that levelof swelling throughout the entire incubation period. Also the presenceof the peptide increases the water absorption of the polymer from about3% by weight to 10% by weight (FIG. 8). Samples of PLA and PCLcontaining identical loading of peptide to the poly(DTH adipate) alsoabsorbed with water within that range during the 2-3 hours that theywere incubated (FIG. 7).

The Effect of the Peptide on the Molecular Weight of the Polymer

One of the amino acid residues on the peptide is an aspartic acid.Aspartic acid is a moiety that introduces acidity into the polymer whenthe polymer is blended with the peptide. Consequently, an investigationof the molecular weight degradation of the polymer was made and comparedto rate of degradation for the neat poly(DTH adipate) (FIG. 9).

As an additional control, samples composed of a blend of 10% (w/w) PEGand 90% (w/w) poly(DTH adipate) were included in these studies becausethese samples absorb 20% by weight water as measured by the TGA. Thisrepresents more water than is absorbed by the polymer samples containing30% (w/w) peptide and functions as a control for the effect of the addedwater on the molecular weight degradation of the polymer. The results ofthese studies were that the samples containing peptide did degrade at afaster rate than the samples that did not contain peptide. After aperiod of over 2 months the poly(DTH adipate) samples containing 30%(w/w) peptide had undergone 40% molecular weight degradation. Incontrast, those samples without peptide demonstrated almost nodegradation during this time period.

In addition, the increased amount of water in the polymer matrix did notaffect the rate of molecular degradation at all. There did not appear tobe any significant difference in the rate of molecular weightdegradation between the poly(DTH adipate) samples containing PEG and theneat samples. It was the presence of the peptide that had the catalyticeffect on the degradation of the polymer. However, this increase indegradation rate was not significant enough to affect the release of thepeptide.

The glass transition temperature of neat poly(DTH adipate) was comparedto those of poly(DTH adipate) containing 15, 20, of 30% (w/w) peptide.The results indicated that the peptide did not reduce the glasstransition temperature of the polymer. In fact, in every case wherepeptide was present the glass transition temperature was higher relativeto the neat polymer samples. The fact that there is an effect on theglass transition temperature indicates that there is a mixing on themolecular scale between the peptide and the polymer. The increase inT_(g) with the addition of the peptide confirms that there is hydrogenbonding between the peptide and the polymer.

Effect of Ionic Strength of the Medium on the Release of the Peptide

Poly(DTH adipate) films containing 30% (w/w) peptide were prepared inthe standard manner. The pH of the incubation media remained about 7,but the ionic strength of the release media was varied. The in vitrorelease of the peptide in HPLC water, in the standard PBS solution (10mM phosphate buffer saline, 138 mM NaCl, 2.7 mM DCl), and in PBS bufferformulated at twice the concentration (20 mM phosphate buffer saline,276 mM NaCl, 5.4 mM KCl) was measured and compared (FIG. 10). It wasobserved that the rate of release of peptide was four times greater inHPLC water as compared to the release rate in phosphate buffer. Theseresults suggest some hydrophobic inter-actions between the peptide andpolymer. Most likely the source of these hydrophobic forces is the pistacking of the tryptophan ring of the peptide with the phenolic ring ofthe polymer.

Incubation of Poly(DTH Adipate) Films Containing 30% (w/w) Peptide inAcidic and Low Ionic Strength Conditions

Samples containing 30% (w/w) peptide were prepared under standardconditions and incubated in HPLC water containing 0.1% (v/v)trifluoroacetic acid, the pH of this solution was 2.2. The release rateof the peptide in this study, where both pH and ionic strength of theincubation media are lowered was greater (FIG. 11) than when just onefactor ws lowered. When just the pH was lowered, 12% of the loadedpeptide was released within three days. When just the ionic strength wasreduced 8% of the loaded peptide was released within three days. Whenboth parameters were lowered simultaneously 20% of the loaded peptidewas released within this time period. Despite enhanced release in theseconditions, the peptide was not “dumped out” as in the case of D.L-PLAbut there was a continuous diffusion of the peptide from the poly(DTHadipate) matrix.

However, what was unexpected was the absorption of water under theseconditions (FIG. 12). Within the first day of incubation these samplesswelled three times relative to the samples incubated in the standardPBS solution, and by the seventh day these samples swelled by seventimes. From FIG. 8, it can be determined that the neat polymer, byitself, does not increase its absorption of water during this initial 7day time period when incubated in the standard PBS solution. Moreover,since this polymer is relatively hydrophobic, as determined from contactangle experiments, it would not be expected that the change inincubation conditions would promote such an increase in the percentageof water uptake by the neat polymer. Therefore, it can be inferred thatit is the peptide within the matrix that is the source of this largewater uptake.

Therefore, incubation in the standard PBS solution favors theinteraction of the peptide with poly(DTH adipate) rather then withwater, hence, there was no increase in swelling beyond the initial 10%even over many weeks of incubation in these conditions. However, inconditions where the peptide-polymer interactions were weakened, as inthis case, where both the pH and ionic strength of the incubation mediawere lowered, there is more of a driving force for the peptide tointeract with water and consequently, there was a steady increase in theswelling of the film as more peptide molecules were exposed andinteracted with water.

Under these conditions of increased acidity and lowered ionic strengththe film samples also turned opaque immediately. This opaqueness, notedonly under the circumstances where there was enhanced release of thepeptide from the poly(DTH adipate) films, appears to be correlated withincreased water absorption by the film samples. The weakening of theintensity of the peptide-polymer interactions result in an increase inwater absorption and the developing opacity, is caused by the water thatoccupies the free volume within the polymer matrix.

The absorption of 10% by weight water was sufficient to release thepeptide to completion in the case of the aliphatic polymers. However,samples whose matrix was composed of poly(DTH adipate) instead of PLA,absorbed 70% by weight water and yet not release the peptide in the same“dumping” manner that the PLA and PCL matrices did at 10% by weight ofwater absorption.

The interaction of the peptide with the tyrosine-derived polyarylatearises from the unique structure of the polymer in which the amide bondof each repeat unit is in close proximity to the pendent ester in thesame unit. This entire region can be considered as one functional group,the α-amidocarboxylate group and can act as a pocket for the hydrogenbonding of various groups on the peptide.

Peptide-Polymer Interactions with Other Tyrosine-Derived Polymers

Several other polymers were screened for the diffusion of the peptide.The loadings of peptide used in these screening experiment were lowerthan those used with poly(DTH adipate) but were sufficient to expectrelease of this readily water-soluble peptide barring any interactionsto impede it.

Poly(DTH dioxaoctanedioate) was the first alternate but structurallyrelated polymer that was investigated. This polymer, contains the DTHrepeat unit which makes it similar to poly(DTH adipate). However, thispolymer is synthesized by polymerizing DTH with dioxaoctanedioic acidinstead of adipic acid. The objective of this experiment was to observethe effect of a more hydrophilic tyrosine-derived polymer on thediffusion of the peptide. It would be expected that this compound ismore hydrophilic than adipic acid because there are two oxygens in thebackbone spacer.

No peptide was released from these films (FIG. 15) indicating thatincreasing the hydrophilicity of the polymer does not have an effect onthe release of the peptide. The water uptake of these films was alsomeasured and found to be 5% by weight in the case of those films thatcontained 10% (w/w) peptide and 10% by weight in the case of those filmscontaining 20% (w/w) peptide. This indicates that although the loadingof the peptide is lower in these specimens there is the same amount ofwater in the bulk in poly(DTH adipate) specimens containing 30% (w/w)peptide as in poly(DTH dioxaoctanedioate) containing 20% (w/w) peptide.In addition, the structure of this polymer differs from poly(DTHadipate) only in the structure of the flexible backbone unit. Since therelease behavior of this polymer is similar to that of poly(DTHadipate), and the structural differences between the two polymers lieonly in the structure of the backbone spacer, it can be concluded thatmost likely it is the DTH unit that is most integral to thepeptide-polymer interactions.

To confirm this last conclusion, another polymer structure wassubstituted for poly(DTH adipate). It was thepoly(DTE_(0.95)-co-PEG_((1000) 0.05) carbonate). This polymer is arandom copolymer of desaminotyrosyl tyrosine ethyl ester (DTE) andpoly(ethylene glycol) (PEG). This copolymer shares the basicdesaminotyrosyl tyrosine alkyl ester repeat unit with the poly(DTHadipate), but contains carbonate linkages and not ester in the backbone,and no diacid component. The absence of the diacid component and thesimilarity in the tyrosine-derived repeat unit should further confirmthat it is the tyrosine-derived component and not the diacid that isinvolved in these interactions should the peptide fail to diffuse fromthis polymer, also. It was also postulated that since PEG is a veryflexible molecule that can hydrogen bond and the length of the PEG islonger than the length of the diacid in the polyarylates, the PEG itselfmight interrupt the hydrogen bonds and allow release of the peptide.Films containing 10% (w/w) peptide were prepared.

Peptide release from these polymers also was minimal. The water uptakeof these samples was also measured, during the period of incubation thefilm samples absorbed 10% by weight water. Again, this is the sameamount of water absorbed by the PLA, PCL, and poly(DTH adipate) samples.Although the peptide loading is lower in these films it is notsurprising that the water uptake is as much as samples of these otherpolymer systems since the PEG increases the hydrophilicity of thesesamples. It appears from this data and the previous data illustratingthe minimal release of the peptide from polymers containing the DTR unitthat the tyrosine-derived repeat unit as suggested above is thestructure responsible for the absence of diffusion of the peptide fromthe polymer.

Poly(DTE carbonate) (FIG. 16) was also formulated with 15% (w/w)peptide. This polymer structure contains only thedesaminotyrosyltyrosine ethyl ester with carbonate linkages and does notcontain any PEG. These films also showed the same behavior as thetyrosine-derived polyarylates (FIG. 17). The water uptake of these filmswas also measured and found to be 6% by weight over the incubationperiod.

Pulsatile Delivery of a Model Water Soluble Peptide Using NovelSynthetic Copolymers

A monomer containing a free acid group randomly replaced the DTH monomerat particular molar concentrations. The particular monomer containingthe free acid group is desaminotyrosyltyrosine (DT) (FIG. 18). Threesets of films from this terpolymer were prepared each set with adifferent molar concentration of DT. The first set waspoly(DT_(0.05)-co-DTH_(0.95) adipate). Since the DT content is theparameter that controls the rate of development of acidity within thesepolymers, the objective of these experiments was to observe the effectof increasing DT content of the polymer on the release of the peptide.

Degradation Mechanism of the Poly(DT-co-DTH Adipate) Polymers

The degradation of the tyrosine-derived polyarylates proceeds via anacid hydrolysis mechanism that is similar to the hydrolysis of poly(DTEcarbonate). The pendent ester groups in contact with water wouldhydrolyze initially and the resulting acid groups would begin thehydrolysis of the backbone ester, liberating DTH and adipic acid. Theadipic acid contributes to the acidity within the matrix and furtherpromotes the hydrolysis of both backbone and pendent ester groups.However, it has been demonstrated that this is a relatively slowprocess, only 40% degradation occurs during a 2 month degradation periodand the degradation rate begins to plateau after reaching this extent ofdegradation (FIG. 9).

The addition of DT to the polymer backbone accelerates the degradationprocess. The degradation rate is hastened because the hydrolyzed pendentester is already present and randomly scattered throughout the polymerprepared for the random scission for the polymer chains. Moreover, sincethe degradation products of the terpolymer are more acidic than those ofpoly(DTH adipate) due to the increase in concentration of DT relative toDTH there is an autocatalytic effect similar to what has been observedwith PLA/PGA derived polymers.

An investigation comparing the effect of increasing mole percent of DTon the degradation rate of poly(DTH adipate) was made (FIG. 19). It wasobserved that with the addition of anywhere between 5 and 15 molepercent DT of sample films had chemically degraded by 90-95% within 3months. These data indicate that DT does catalyze degradation, but italso indicates that there is a maximum limit to the catalytic effect ofthe DT. This is seen in the similarity in rate of degradation betweenthe polymer containing 10 mole percent of DT and that with 15 molepercent of DT.

In addition, it can also be concluded from the percent molecular weightretention data that the autocatalytic effect is present in thesepolymers. This can be observed in the rate of degradation of the polymerwith 5 mole percent DT. The rate of degradation begins much slower thanthe rate of degradation of the polymers with higher percentages of DT.However after 120 days the percent molecular weight retention isapproximately the same as it is for the samples with higher DT content.This suggests that whatever initial acidity developed in the matrix ofthe polymer containing 5 mole percent DT resulted in further increasedhydrolysis of the pendant chain of the DTH repeat unit, converting theDTH repeat unit to the DT repeat unit. In this manner, the number of DTrepeat units increased from the original 5 mole percent andconsequently, the rate of degradation of this polymer increased until itwas able to “catch up” with the rate of degradation of the polymerscontaining higher mole percentages of DT.

Water Uptake of Poly(DT-co-DTH Adipate) Films

In spite of the increased hydrophilicity of DT versus DTH there is nosignificant effect of mole percent of DT on amount of water absorbed bythe polymer when samples are initially incubated (FIG. 20). Moreoversignificant water uptake does not appear to cause degradation, rather itseems to be an effect of the degradation. This can be observed bycomparing the water uptake during the first two weeks of incubation.During these two weeks the rate of degradation is highest fro thepolymers containing 10 or 15 mole percent DT, yet this is also theperiod of time that is associated with the lowest percent water uptake.This is further observed by noting that the polymers that do absorb morethan 20% water do not absorb this until they have degraded by 90%.

Visual Inspection of Poly(DT-co-DTH Adipate) Containing Peptide

The Three types of terpolymers were formulated in the same technique asused for the copolymer, poly(DTH adipate), and two loadings of peptidewere explored. One loading was 15% (w/w) peptide and the other 30% (w/w)peptide. Those films containing 15% (w/w) peptide were completelytransparent, there was not difference between the neat copolymer filmsand films that contained 15% (w/w) peptide. Samples from films thatcontained 30% (w/w) were slightly hazy. All of the polymers wereflexible and easy to handle.

Analysis of Miscibility of Terpolymer Using DSC

Thermograms of these terpolymers indicated only one glass transitionwhich was in the vicinity of the glass transition of poly(DTH adipate).The appearance of only one glass transition indicates there is amiscibility between the DTH adipate and the DT adipate, not surprisingsince they share a very similar structure. Moreover, the range oftemperatures over which the glass transition occurs is about 6° C. Thisis about the same for poly(DTH adipate) indicating that the polymer isquite homogeneous.

There was a trend of increasing T_(g) with increasing mole percent ofDT, this is quite expected since an increase in the amount of DT couldresult in an increase in hydrogen bonding between the chains and therebyincreasing the rigidity of the polymer (Table 1). The homogeneity of thecopolymer, in all probability, contributes to the transparency andclarity of those films that contain peptide.

TABLE 1 T_(g) of poly(DT_(x)-co-DTH_(1−x)adipate)x = 5, 10, 15 MolePercent DT in Polymer T_(g)(° C.) 0 37 5 37 10 43 15 46Peptide Release from Terpolymer Films Containing 15% (w/w) Loading

The incubation conditions were the same used in the above experiments.The results of these experiments were a delayed release, and the lengthof the delay time was a function of the mole percent of DT. The set offilms containing 15 mole percent DT was characterized by a lag time of20 days, after this lag time, 60% of the loaded peptide was releasedover a period of 40 days (FIG. 21). Samples containing 10% DT wereassociated with a lag time of close to 60 days. This delay period wasfollowed by a release phase where 60% of the loaded peptide was releasedwithin 30 days. Samples with 5% DT never released the peptide even after110 days of incubation. The control in this experiment was poly(DTHadipate) samples containing 15% (w/w) peptide which, also, did notrelease the peptide. In all samples no burst was observed and noleaching of the peptide occurred during the lag time.

The correlation of shorter lag time with increasing mole percent of DTis not unexpected. The samples containing larger mole percents of DTwould be expected to accumulate acidic degradation products fastercreating a higher concentration of these products in the bulk of thepolymer resulting in the weakening of the peptide-polymer interactionsearlier than those polymers with lower percentages of DT.

Peptide Release from Terpolymer Films Containing 30% (w/w) Peptide.

In contrast to the terpolymer samples containing 15% (w/w) peptide thatwere not characterized by a burst of peptide, these samples whichcontained 30% (w/w) all demonstrated large bursts (FIG. 22). The burstswere proportional to the mole percent of DT in the polymer. The reasonfor the large burst observed with the higher peptide loading could bethe following: since the pK_(a) of the tyrosine acid proton isapproximately 2, it would be expected that when incubated initially, themajority of DT acid protons would be lost to the medium. Therefore, anypeptide molecules that would be hydrogen bonded to this proton would nolonger be interacting with this group once the proton is lost, andtherefore these peptide molecules would be lost as a burst. In addition,after the loss of the acid proton, the carboxyl-ate group of the DTmight actually compete with the peptide for interaction sites on the DTHrepeat unit resulting in the release from the films of the peptidemolecules that lost the competition. Furthermore, the higher the DTcontent in the polymer the more competition for the peptide andconsequently, the size of the burst is correlated with increasing molepercent of DT. However, following the initial changes that occur whenthe specimens are first incubated a new equilibrium is established ofall the components, and no further release of peptide occurs for manydays. This phenomenon is not observed with those terpolymer samplescontaining the lower loading of peptide because there are many fewermolecules of peptide and sufficient DTH sites for both the interactionof the peptide and the DT.

Samples containing 10% DT were also characterized by a second releasephase. This second release phase occurred at approximately 60 days whichis also when the release of the peptide ws observed in samples of thispolymer containing 15% (w/w) peptide. This release is due to theweakening of the interactions associated with the drop in pH of thepolymer matrix. This polymer is quite unique among this grouping becausethese specimens alone can be considered a pulsatile release system. Thefilms containing 15% DT also demonstrated a second release phase atabout 40 days but it is much smaller than the second release phase ofthe specimens containing 10% DT. Samples with 5% DT, again as in the 5%DT samples containing 15% (w/w) peptide, presumably, never reached thecritical pH necessary for release of the peptide, and, therefore,following the burst no more peptide was released.

Analysis of the Buffer Media of the Poly(DT-co-DTH Adipate) Containing15% (w/w) Peptide for pH Changes

The buffer media were analyzed for pH changes at each buffer change(FIG. 23). Since the release of the peptide depends on the lowering ofthe pH of the matrix a detectable lowering of the pH should coincidewith the release of the peptide. As expected, those films composed ofthe polymer system with 15 mole percent of DT demonstrated a drop of thepH below 7.2, first. This reduction in pH began at approximately 30 dayswhich was 10 days after release of the peptide commenced. The pH of themedia remained around 7.0 for the remainder of the incubation.

Samples containing 10 mole percent of DT were characterized by a drop inpH below 7.2 beginning around 60 days, which is precisely when releaseof the peptide commenced. The pH of the media of these samples remainedapproximately 7.0 for the remainder of the incubation period.

Specimens containing 5 mole percent of DT behaved exactly like thepoly(DTH adipate) samples that did not contain any DT at all. Both typesof samples remained between 7.3 and 7.4 for the first 100 days of theincubation. After this time both types of samples dropped to andremained at 7.2 for the remainder of the incubation period. The dataindicate that there is a correlation between release of the peptide andgeneration of acidic degradation products. Specifically, only thosesamples that released peptide were associated with a drop in pH below7.2 and this drop coincided with peptide release.

In addition, control samples of poly(DTH adipate) containing 15% (w/w)peptide were placed in buffer at pH of 7.0. This again, was to observewhether environmental pH affects the release of the peptide. Tracerelease of the peptide was seen from these control samples. Nodifference in the behavior of these samples as compared to samplesincubated in buffer at 7.4 was observed.

Chemical Integrity of the Released Peptide from Poly(DT-co-DTH Adiapte)Matrices

The only polymer matrix of the group of polymers investigated in theseexperiments that released any of the peptide with the cleaved disulfidebond was the poly(DT_(0.15)-co-DTH_(0.85) adipate) samples whichcontained the lower loading of peptide. These samples began the releasephase after a lag time of 20 days and continued this steady releaseuntil approximately 60 days of incubation. Intact peptide was releasedwithin the first 20 days of the release phase. However from the 44^(th)day of incubation and beyond, fully one third of the peptide releasedwas associated with a cleaved disulfide bond. Again, peptide with acleaved disulfide bond was not observed in association with any otherpolymer system in these studies.

Percent Molecular Weight Retention During In Vitro Incubation ofPoly(DT-co-DTH Adipate) Samples Containing 15% (w/w) Peptide

The percent molecular weight retention data of the various sets of filmscontaining 15% (w/w) peptide were not significantly different thansamples without peptide (FIGS. 24 and 19). This suggests that thecatalytic degradation effect on the polymer of the DT is more importantthan the catalytic effect of the aspartic acid group of the peptide. Inaddition, although the polymer containing 15% DT released the peptidefar earlier than the polymer with 10% DT the molecular weightdegradation rate was the same. The explanation for this observation isthat there is a sufficient number of DT repeat units in both polymersystems to reach the maximum rate of hydrolysis. However due to theincreased amount of DT in the poly(DT_(0.15)-co-DTH_(0.85) adipate)relative to the polymer with 10 mole percent DT, the degradationproducts also contain more DT and therefore critical concentration ofacidic products necessary for release of the peptide is reached earlierwith these samples than the polymers with 10 mole percent of DT.

Comparison of the Mechanism of Degradation of the Poly(DT-co-DTHAdipate) Samples with and without Peptide

The poly(DT-co-DTH adipate) polymers without peptide appear to degradethrough the same mechanism. The reates may be different especiallybetween those polymers that contain 5 mole percent of DT and those thatcontain more DT but the end result appears similar. After 16 weeks ofincubation the polymers have all developed a significant amount of lowmolecular weight fractions and there does not appear to be a preferencefor the formation of one particular fraction over another.

In contrast, the samples that were formulated with peptide do not alldegrade by the same mechanism. Samples with 5 mole percent DT behavesimilarly to the neat samples; there is the random scission of thechains forming a variety of oligomers and no special preference for theformation of a specific degradation product is observed in the GPCchromatograms of the degraded samples. This observed behavior wasconsistent for this polymer system whether it was loaded with 15% (w/w)peptide, 30% (w/w) peptide or neat.

However, those films of poly(DT_(0.10)-co-DTH_(0.90) adipate) containingpeptide (it was the same for both loadings of peptide) exhibited adistinct preference for the formation of monomer during the degradationprocess. The monomer (DTH) has a retention time of 18.6 minutes in theGPC and starting from the 5^(th) week of incubation and beyond there isthe presence of a well defined peak at this retention time in thechromatograms of these samples. This implies that the polymer degradesin the random scission manner until it reaches about 20% molecularweight retention at five weeks of incubation. After this point thepolymer begins to degrade in an unzipping process. This unzippingprocess means that the degradation begins from the chain ends and movesin along the chain. Consequently, each scission results in the formationof monomer. The poly(DT_(0.15)-co-DTH_(0.85) adipate) samples thatcontain peptide exhibited this same behavior as described for thepoly(DT_(0.10)-co-DTH_(0.90) adipate) samples that contain peptide.

Physical Disintegration During In Vitro Incubation of Samples ofPoly(DT-co-DTH Adipate) Samples Containing Peptide

There were no significant physical changes in these samples for thefirst two weeks of incubation. However, by the third week all thesamples have become opaque, and by the fourth week there was significantshredding of the samples containing 15% DT. IN fact, so much shreddinghas occurred that the buffer media has turned opaque. No significantshredding of the samples containing 10% DT occurred before 70 days andthe samples containing 5% DT never shredded at all.

This shredding is most likely related to the dissolution of the watersoluble degradation products of the polymers. Other polymers with thehigher free acid content contain a significant amount of water solubledegradation products (DT and adipic acid) and therefore shredding iscommon to both of them. Since shredding occurs in both of these polymersystems the resulting films following incubation of 80 or more daysappeared transparent with only a “skin” of the material left. All of thebulk had disappeared. Shredding never occurs in the polymer with thelower free acid content since it never develops enough water solublecomponents within the bulk and therefore, though, these samples swelledand curled during the incubation period they remained smooth and intact.The same phenomena was observed with the poly(DT-co-DTH adipate) samplescontaining 30% (w/w) peptide.

The foregoing illustrates that polymers that form hydrolytic degradationprecuts promote the release of biologically active compounds from thepolymer matrix in comparison to polymers of similar structure to do nothydrolytically degrade. Neither polymer initially releases thebiologically active compound. However, a delayed pulsatile release isobtained from polymers that hydrolytically degrade as the degradationprecuts accumulate, while significant quantities of biologically activecompound are never released from the polymers that do not hydrolyticallydegrade.

The foregoing examples and description of the preferred embodimentshould be taken as illustrating, rather than as limiting, the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not to be regarded as a departure fromthe spirit and scope of the invention, and all such modifications areintended to be included within the scope of the following claims.

1. An implant comprising: a weight percentage of a peptide drug having achemical structure with hydrogen bonding sites; and a hydrolyticallydegradable polycarbonate copolymer having a molar percentage oftyrosine-derived diphenol monomer units with pendant carboxylic acidgroups and a molar percentage of tyrosine-derived diphenol monomer unitswith pendant carboxylic acid ester groups, wherein the peptide drug isdispersed within the copolymer.
 2. The implant of claim 1 wherein thepolycarbonate copolymer degrades hydrolytically promoting release of thepeptide drug from the implant.
 3. The implant of claim 1 wherein themolar percentage of tyrosine-derived diphenol monomer units with pendantcarboxylic acid groups is between about 5 mole percent and about 15 molepercent.
 4. The implant of claim 1 wherein the weight percentage of thepeptide drug is between about 15 weight percent and about 30 weightpercent.
 5. The implant of claim 1 wherein the molar percentage oftyrosine-derived diphenol monomer units with pendant carboxylic acidgroups and the weight percentage of the peptide drug is effective toprovide reproducible release profiles of the peptide drug from theimplant without an initial burst effect.
 6. The implant of claim 1wherein the copolymer is a desaminotyrosyltyrosine copolymer ofpoly(desaminotyrosyltyrosine ethyl ester carbonate).
 7. The implant ofclaim 1 wherein the peptide drug is a cyclic peptide.
 8. A formulationcomprising: a weight percentage of a biologically active compound havinga chemical structure with hydrogen bonding sites; and a hydrolyticallydegradable polycarbonate copolymer having a molar percentage oftyrosine-derived diphenol monomer units with pendant carboxylic acidgroups and a molar percentage of tyrosine-derived diphenol monomer unitswith pendant carboxylic acid ester groups, wherein the polycarbonatecopolymer degrades hydrolytically promoting release of the biologicallyactive compound from the formulation.
 9. The formulation of claim 8wherein the molar percentage of tyrosine-derived diphenol monomer unitswith pendant carboxylic acid groups and the weight percentage of thebiologically active compound is effective to provide reproduciblerelease profiles of the biologically active compound from theformulation without an initial burst effect.
 10. The formulation ofclaim 8 wherein the biologically active compound is a pharmaceuticallyactive compound.
 11. The formulation of claim 10 wherein thepharmaceutically active compound is a peptide.
 12. The formulation ofclaim 11 wherein the peptide is a cyclic peptide.
 13. The formulation ofclaim 8 wherein the copolymer is a desaminotyrosyltyrosine copolymer ofpoly(desaminotyrosyltyrosine ethyl ester carbonate).
 14. The formulationof claim 8 wherein the molar percentage of tyrosine-derived diphenolmonomer units with pendant carboxylic acid groups is between about 5mole percent and about 15 mole percent.
 15. The formulation of claim 8wherein the weight percentage of biologically active compound is betweenabout 15 weight percent and about 30 weight percent.
 16. A method fordelayed delivery of a peptide drug to a patient in need thereofcomprising: providing a formulation including the peptide drug and acopolymer, wherein the peptide drug has a chemical structure withhydrogen bonding sites, and wherein the copolymer is a hydrolyticallydegradable polycarbonate copolymer having tyrosine-derived diphenolmonomer units with pendant carboxylic acid groups and tyrosine-deriveddiphenol monomer units with pendant carboxylic acid ester groups; andadministering the formulation to the patient so that release of thepeptide drug occurs after a predetermined time without an initial bursteffect.
 17. The method of claim 16 wherein a weight percentage of thepeptide drug in the formulation is between about 15 weight percent andabout 30 weight percent.
 18. The method of claim 16 wherein a molarpercentage of tyrosine-derived diphenol monomer units with pendantcarboxylic acid groups is between about 5 mole percent and about 15 molepercent.
 19. The method of claim 16 wherein the peptide drug is a cyclicpeptide.
 20. The method of claim 16 wherein the copolymer is adesaminotyrosyltyrosine copolymer of poly(desaminotyrosyltyrosine ethylester carbonate).